Clostridial neurotoxins (tetanus and seven serotypes of botulinum neurotoxin) each block vesicular neurotransmitter release by cleaving specific proteins implicated in synaptic vesicle fusion. These toxins are important therapeutic agents for a number of neurologic disorders including cerebral palsy and are valuable tools for understanding neurotransmitter release, membrane trafficking, and protein sorting, transport, and targeting. We have studied the presynaptic action of the toxins using biochemical and morphologic techniques in primary cell cultures of fetal mouse spinal cord. We have found that the drug fumonisin B1, which blocks ganglioside synthesis, protects neurons against the action of botulinum neurotoxin A (BoNT A) and that addition of exogenous gangliosides restores toxin action. In this regard, GQ1b and GT1b are approximately equivalent and GD1b is less effective. These findings indicate that neuronal membrane gangliosides are required for delivery of BoNT A to the neuronal cytosol. Both A and E serotypes of BoNT cleave the neuronal protein SNAP-25. Whereas the ensuing blockade of neurotransmitter release is consistent with the amount of BoNT E-damaged SNAP-25, block of release is almost twice as great as that expected from the levels of BoNT A-damaged SNAP-25. We have obtained evidence that the BoNT A cleavage product of SNAP-25 acts as a competitive inhibitor of intact SNAP-25, and blocks synaptic vesicle fusion in intact neurons at physiological concentrations of calcium. This finding is consistent with the greater potency of BoNT A relative to BoNT E.